1. Field of the Invention
A method is provided for identifying agonists and antagonists of dipeptidyl peptidase I in a high-throughput format. The method uses a peptide substrate that is label-free and binds to multiple substrate binding sites of dipeptidyl peptidase I.
2. Description of the Related Art
Dipeptidyl peptidase I (DPPI) or cathepsin C is a member of the lysosomal papain-type cysteine protease family that also includes cathepsin B, K, H, L, O, and S (Bouma and Gruber, 1996, Biochem. Biophys. Acta 113: 350-358; Turk et al., 2000, Biochem. Biophys. Acta 1477: 98-111). This protease family activates serine proteinases in immune and inflammatory cells.
The physiological role of DPPI is to convert inactive proenzymes into active enzymes by removing two amino acids as a dipeptide unit from the N-terminal end of the proenzymes in immune and inflammatory cells (reviewed by Caughey, G. H., 2002, Molecular Immunology 38: 1353-1357; Wolters et al., 2001, J. Biol. Chem. 276: 18551-18556; McGuire et al., 1993, J. Biol. Chem. 268: 2458-2467; Pham and Ley, 1999, Proc. Natl. Acad. Sci. 96: 8627-8632; Pham et al., 2004, J. Immunol. 173: 7277-7281). DPPI activates many serine proteinases including chymase, tryptase, cathepsin G, elastase, and neutrophil-elastase, granzymes A and B from T-lymphocytes and natural killer cells; and rheumatoid arthritis proteases (Adkison et al., 2002, J. Clin. Invest. 109: 363-371). The enzymes or serine proteases activated by DPPI are needed for defense responses. In addition, the unregulated DPPI has been shown to cause over-activation of proteases associated with diseases including chronic obstructive pulmonary disease, Papillon-Lefevre Syndrome, Sepsis, arthritis and other inflammatory disorders. Therefore, DPPI is a potential target for therapeutic treatment.
It is proposed that DPPI consists of four identical monomers. Each monomer is produced from a precursor polypeptide, which is cleaved into a N-terminal exclusion domain, an activation peptide, and a papain-like domain. The papain-like domain is further cleaved into a heavy chain and a light chain. Subsequently, the N-terminal exclusion domain, the heavy and the light chains fold into a monomer, which tetramizes into the mature DPPI of about 200 kDa (Turk et al., 2001 EMBO J., 20: 6570-6582).
Recent crystallization of DPPI identified some structures, including an active site cleft and substrate binding sites, involved in the dipeptidyl proteolytic activity. The substrate binding sites reside on the external part of the papain-like structure and form hydrogen bonds with the substrates. The site that binds to the first amino acid at the N-terminal side of the cleaved bond of the substrate is referred to as the S1 binding site, the site that binds to the second amino acid at the N-terminal side is referred to as the S2 binding site. Further, the site that binds to the first amino acid at the C-terminal side of the cleaved bond of the substrate is referred to as the S1′ binding site, and the site that binds to the second amino acid at the C-terminal side is referred to as the S2′ binding site, etc (Turk et al., 2001, EMBO J., 20:6570-6582; Molgaard et al., 2007, Biochem. J., 401:645-650). The corresponding site in a DPPI substrate is referred to as P1, P2, P1′, P2′ etc, where P1 of the substrate binds to the S1 site of DPPI, P2 of the substrate binds to S2 of DPPI, and P1′ of the substrate binds to the S1′ site of DPPI, etc. Based on the crystallographic results of a binding complex of DPPI and a substrate of 6 amino acids, Molgaard et al. proposes a model where DPPI contains the S2-S1 and S1′-S4′substrate binding sites. Since DPPI binds to larger substrates such as human prochymase of 228 amino acids in vivo; the substrate binding sites of DPPI may contain more than S2-S1-S1′-S4′, even to S2-S1-S1′-S18′.
As DPPI is a potential therapeutic target, several assays for analyzing DPPI activity have been commonly used (Gelman et al., J. Clin. Invest. 1980, 65: 1398-1406; (Tran et al., Arch. Biochem. Biophys. 2002, 403: 160-170). Gelman et al. uses a labeled dipeptide of Gly-Phe-β-naphthylamide as substrate and determined DPPI activity by measuring the concentration of free β-naphthylamide with a spectrophotofluorometer. Similarly, Tran et al. uses several dipeptides labeled with 7-amino-4-methylcoumarin as DPPI substrates and determined DPPI activity by measuring the concentration of free methylcoumarin with a spectrophotofluorometer. Tran et al. shows that the dipeptide substrate of Ala-Hph-7-amino-4-methylcoumarin is the best substrate for DPPI, even though the substrate contains a non-physiological residue Hph (homophenylalanine).
These commonly used dipeptide substrates with fluorescence labels have several problems. First, the dipeptide substrate only binds to the S1 and S2 sites. This is partial or incomplete in comparison to biological substrates in vivo which binds to a binding site in addition to the S1 and S2 sites. The incomplete or partial binding may be non-specific and hence lead to the identification of false positive or negative compounds. Also, the additional step of fluorescence detection is needed to detect the DPPI activity. This additional step makes the assay difficult to be adapted for use in a high-throughput format.
Therefore, there is still a need to develop an assay that uses a substrate which is label-free and biologically related for analyzing DPPI activity. Also, there is a need to develop a DPPI assay suitable for use in a high-throughput format to enable efficient screening of a large number of compounds or agents for drug development.
The label-free technology has been combined with a mass spectrometry system to analyze enzymatic reactions. Quercia et al. (J. Biomol. Screen. 12: 473-480, 2007) identifies the kinase AKT1/PKBα inhibitors using mass spectrometry in multiple reaction monitoring. Similarly, Forbes et al. (J. Biomol. Screen. 11: 628-634, 2007) evaluates phosphortidylserine decarboxylase in 96-well plates using mass spectrometry in multiple reaction monitoring. A mass spectrometry detection system in a high-throughput format has not been applied for analyzing DPPI activity.
The present application provides a method for assaying DPPI activity. The method utilizes a label-free peptide substrate that comprises biologically related amino acid sequences and binding specificity. The assay may be detected using a mass spectrometry system and may be operated in a high-throughput format, which reduces processing time and increases throughput which are desired for screening compounds. The method is also useful for differential tracking of fragment-based compounds for drug development and evaluating other dipeptidyl proteolytic reactions for functional studies.